International Journal of Systematic and Evolutionary Microbiology (2011), 61, 40–44
DOI 10.1099/ijs.0.019638-0
Streptococcus ursoris sp. nov., isolated from the
oral cavities of bears
Noriko Shinozaki-Kuwahara, Kazuko Takada and Masatomo Hirasawa
Correspondence
Kazuko Takada
Department of Microbiology and Immunology, Nihon University School of Dentistry at Matsudo,
Matsudo, Chiba 271-8587, Japan
[email protected]
Three Gram-positive, catalase-negative, coccus-shaped organisms were isolated from the oral
cavities of bears. The isolates were tentatively identified as a streptococcal species based on the
results of biochemical tests. Comparative 16S rRNA gene sequencing studies confirmed that the
organisms were members of the genus Streptococcus, but they did not correspond to any
recognized species of the genus. The nearest phylogenetic relative of the new isolates was
Streptococcus ratti ATCC 19645T (98.6 %), however, DNA–DNA hybridization analysis showed
that the isolates displayed less than 15 % DNA–DNA relatedness with the type strain of S. ratti.
Colonies of the novel strains grown on mitis salivarius agar showed an extracellular
polysaccharide-producing colony morphology. Based on phenotypic and phylogenetic evidence,
it is proposed that the novel isolates are classified in the genus Streptococcus as Streptococcus
ursoris sp. nov. The type strain of S. ursoris is NUM 1615T (5JCM 16316T5DSM 22768T).
The genus Streptococcus embraces a broad range of Grampositive, facultatively anaerobic, catalase-negative, chainforming and coccus-shaped organisms. A large number of
streptococcal species colonize the oral cavities of animals
and humans and these oral streptococci have been divided
into five major groups (Facklam, 2002). Among oral
streptococci, the mutans group is responsible for dental
caries (Hamada & Slade, 1980). These bacteria are known
to form characteristic colonies on mitis salivarius (MS)
agar plates demonstrating extracellular polysaccharide
synthesis. In humans, it is proposed that water-insoluble
glucan, which is synthesized by the action of glucosyltransferase (GTF) from sucrose as the substrate, is one of
the most important cariogenic factors (Hamada & Slade,
1980). Several kinds of mutans streptococcal species, which
can synthesize glucans, have been isolated from the oral
cavities of various animals, for example, hamster, rat,
monkey, pig, bat and wild boar (Beighton et al., 1981;
Whiley & Beighton, 1998; Takada & Hirasawa, 2007, 2008,
2010). Bears are known to be omnivorous animals and may
have the chance to ingest sucrose in the form of sweet
fruits, such as berries, and in honey. Dental caries have
been reported in the oral cavities of wild bears (Manville,
1990). In this study, three previously unidentified strains
isolated from the oral cavity of Japanese black bears (Ursus
Abbreviations: GTF, glucosyltransferase; MS, mitis salivarius.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and
groEL gene sequences of strain NUM 1615T are AB501126 and
AB541991, respectively.
Supplementary figures and a supplementary table are available with the
online version of this paper.
40
thibetanus japonicus) and capable of glucan synthesis on
MS agar were characterized.
Examination of the oral microflora of bears was performed
on MS agar (BD Difco). MS agar is widely used to isolate
strains of Streptococcus mutans as well as other oral
streptococcal species. Three streptococci-like strains
(NUM 1610, NUM 1615T and NUM 1619) obtained from
samples from the oral cavities of wild bears were isolated
on MS agar. The isolated strains formed small, raised,
adherent colonies with irregular margins. The cocci stained
as Gram-positive. The strains were grown on brain heart
infusion (BHI, BD Difco) agar supplemented with 5 %
horse blood at 37 uC under an atmosphere of 95 % N2 and
5 % CO2 with non-haemolytic activity. Catalase activity
was found to be negative. The organisms were characterized biochemically using the Rapid ID32 Strep, API 50 CH
and API ZYM systems according to the manufacturer’s
instructions (bioMérieux). The colony formation resembled
that of members of the mutans streptococcus group,
although the biochemical characteristics did not correspond
to any recognized species of the genus Streptococcus. The
new isolates were subjected to further genetic studies.
Serological analyses of isolates were conducted by agar-gel
immunodiffusion methods using rabbit antisera raised
against reference strains of mutans streptococci prepared
previously (Hirasawa et al., 1980; Takada et al., 1984).
Rantz-Randall extract antigens were prepared from cells
cultured overnight on BHI as described previously (Rantz
& Randall, 1955). Immunodiffusion experiments with
isolate NUM 1615T showed that no cross-reacted precipitin
bands formed with any sera prepared against mutans
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Novel oral streptococci from bears
Table 1. Characteristics that differentiate strain NUM 1615T from closely related streptococcal species
Taxa: 1, NUM 1615T; 2, S. ratti ATCC 19645T; 3, S. devriesei CCUG 47155T; 4, S. mutans NCTC 10449T; 5, S. ferus ATCC 33477T; 6, S. macacae
NCTC 11558T. Data for taxa 2, 4, 5 and 6 are from Whiley & Beighton (1998). Data for taxon 3 are from Collins et al. (2004) and the present study.
All taxa give a positive reaction in the Voges–Proskauer test and are positive in tests for the hydrolysis of aesculin and the fermentation of mannitol
and sorbitol. +, Positive; 2, negative; ND; not detected.
Characteristic
Hydrolysis of:
Arginine
Fermentation of:
Inulin
Melibiose
Raffinose
D-Ribose
Starch
Sensitivity to bacitracin (2 U ml21)
Serovar(s)
GTF activity
DNA G+C content (mol%)
Source
1
2
3
4
5
6
+
+
–
–
–
–
–
+
+
+
–
+
+
+
+
–
–
–
b
+
41–43
Rat, human
+
+
+
–
–
+
+
+
+
–
–
–
c, e, f
+
36–38
Human
+
–
–
–
+
+
c
+
43–45
Rat
–
–
+
–
–
+
c
+
35–36
Monkey
ND
+
33–35
Bear
streptococci strains of serotypes a to h, suggesting that
strain NUM 1615T could not be grouped with any known
serotype of mutans streptococci. The Lancefield grouping
test was performed using the Streptococcal grouping kit
(Oxoid). No Lancefield carbohydrate antigens were
detected from strain NUM 1615T. Strain NUM 1615T
was able to synthesize adhesive water-insoluble glucan
from sucrose as the substrate and showed sucrosedependent colonization by the action of GTF enzymes.
The phenotypic characteristics that differentiated the new
strains from closely related streptococcal species are shown
in Table 1.
To determine the G+C contents, DNA–DNA relatedness
and 16S rRNA gene sequences of the isolated strains,
chromosomal DNA was extracted and purified as described
previously (Shiroza et al., 1998). Measurement of the G+C
content of the DNA was carried out by HPLC according to
the method of Hirasawa & Takada (1994). The G+C
contents of the three strains ranged from 33 to 35 mol%
and were similar to those previously determined for
Streptococcus macacae and S. mutans. To assess the
ND
–
42
Horse
phylogenetic position of the newly isolated strains, their
16S rRNA genes were amplified by PCR using primers
described previously by Takada & Hirasawa (2007),
directly sequenced with an ABI PRISM 3130 Genetic
Analyzer using a Big Dye Terminator v1.1 Cycle Sequencing kit (Life Technologies Co.) and then subjected to a
comparative analysis. The closest known relatives of the
new isolates were determined by performing DDBJ/EMBL/
GenBank database searches. The 16S rRNA gene sequences
were compared using BLAST algorithms. Distance matrices
were determined following the assumptions described by
Kimura (1980). Phylogenetic trees were constructed
according to the neighbour-joining method (Saitou &
Nei, 1987) and the topology of the tree was estimated
by bootstrap analysis using the CLUSTAL W program
(Thompson et al., 1994). Highest sequence similarities
were obtained with Streptococcus ratti ATCC 19645T
(98.6 %). The next most closely related strains were
Streptococcus devriesei CCUG 47155T (96.0 %) and S.
mutans NCTC 10449T (95.1 %), both of which were
below the 97 % threshold value for the definition of
separate species. The phylogenetic tree constructed by the
Fig. 1. Phylogenetic tree of members of the
genus Streptococcus inferred from comparison
of 16S rRNA gene sequences by the neighbour-joining method. Bar, 0.01 substitutions
per nucleotide position. An extended version of
this tree is available as Supplementary Fig. S1.
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41
N. Shinozaki-Kuwahara, K. Takada and M. Hirasawa
neighbour-joining method (Fig. 1; an extended version of
this tree is available as Supplementary Fig. S1 in IJSEM
Online) revealed a clear affiliation between strain NUM
1615T and the genus Streptococcus. The phylogenetic trees
constructed by the neighbour-joining and maximumparsimony methods (see Supplementary Fig. S2 in IJSEM
Fig. 2. Phylogenetic tree based on 757 bp of groEL gene fragment sequences constructed by using the neighbour-joining method.
Bootstrap values .50 % calculated for 1000 subsets are shown at branch-points. Bar, 0.01 substitutions per nucleotide position.
42
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Novel oral streptococci from bears
Online) showed a similar evolutionary process for strain
NUM 1615T and S. ratti ATCC 19645T. A phylogenetic tree
(Fig. 2) constructed by the neighbour-joining method with
partial sequences of the groEL (757 bp) gene (Glazunova
et al., 2009) also confirmed the phylogenetic placement of
representative strains within the genus Streptococcus.
DNA–DNA hybridization experiments were performed as
previously described (Takada & Hirasawa, 2007). DNA–
DNA relatedness was examined by labelled DNA from strain
NUM 1615T with unlabelled single-stranded DNA from
related streptococci (see Supplementary Table S1 in IJSEM
Online). The three new isolates showed DNA–DNA relatedness values of .70 % with each other and therefore these
results confirmed that the isolates belonged to the same
species (Johnson, 1973). Since the three isolates exhibited
high degrees of relatedness, their relationship at the strain
level was confirmed. S. ratti ATCC 19645T showed a high 16S
rRNA gene sequence similarity to strain NUM 1615T, but the
DNA–DNA relatedness value between the two strains was
only 13.3 %. The DNA from other streptococci, such as S.
devriesei and S. mutans, showed low levels of DNA–DNA
relatedness to strain NUM 1615T (all ,32 %).
Therefore, based on phenotypic and phylogenetic criteria,
it is considered that strains NUM 1615T, NUM 1610 and
NUM 1619, isolated from wild bears, warrant classification
as a novel species of the genus Streptococcus, for which the
name Streptococcus ursoris sp. nov. is proposed.
Description of Streptococcus ursoris sp. nov.
Streptococcus ursoris (urs.o9ris. L. n. ursus -i bear; L. n. os
oris mouth; N.L. gen. n. ursoris of the mouth of a bear).
Cells stain Gram-positive, are non-spore-forming cocci,
0.5–0.7 mm in diameter and occur in pairs or short chains.
Colonies are white, non-haemolytic and 0.75–1.0 mm in
diameter after incubation on blood agar at 37 uC for 24 h.
On MS agar, colonies appear small, dark blue and crinkled.
Facultatively anaerobic and catalase-negative. Lancefield
carbohydrate antigens are not detected. Acid is produced
from D-glucose, D-fructose, D-galactose, D-mannose, mannitol, sorbitol, N-acetylglucosamine, salicin, cellobiose,
maltose, lactose, sucrose, trehalose, raffinose, D-tagatose
D-ribose and melibiose, but not from D-arabinose, Dxylose, glycogen, inulin or starch. Aesculin is hydrolysed,
but hippurate is not. Voges–Proskauer test is positive.
Enzyme activities are detected for arginine dihydrolase,
alanyl-phenylalanyl-proline arylamidase, b-glucosidase, aglucosidase, a-galactosidase, naphthol-AS-BI-phosphohydrolase, acid phosphatase, valine arylamidase, cystine
arylamidase and leucine arylamidase. b-Galactosidase, bglucuronidase, alkaline phosphatase, N-acetyl-b-glucosaminidase and urease are not produced. Sensitive to
bacitracin. In BHI broth, there is no apparent growth at
45 uC and the GTF enzyme is produced.
The type strain, NUM 1615T (5JCM 16316T5DSM
22768T), was isolated from clinical specimens obtained
http://ijs.sgmjournals.org
from the oral cavity of a bear. The DNA G+C content of
the type strain is 34 mol%.
Acknowledgements
We are grateful to Dr Hans Trüper for suggesting the species name.
This study was supported in part by a Grant-in-Aid for Young
Scientists and a Grant-in-Aid for SBFSR (2008–2012) from MEXT.
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